High hydrostatic pressure treatment for the inactivation of Staphylococcus aureus in human blood plasma

For the past 30years, pressure inactivation of microorganisms has been developed in biosciences, in particular for foods and more recently for biological products, including pharmaceutical ones. In many past studies, the effect of high hydrostatic pressure (HHP) processes on pathogens focused mainly on the effect of an increase of the pressure value. To assure the safety of pharmaceutical products containing fragile therapeutic components, development of new decontamination processes at the lowest pressure value is needed to maintain their therapeutic properties. The aim of this study was therefore to evaluate the impact of the process parameters characterizing high-pressure treatments [such as the pressurization rate (PR) and the application mode (AM)] on the inactivation of pathogens, in particular to determine how these parameters values could help decrease the pressure value necessary to reach the same inactivation level. The effect of these physical parameters was evaluated on the inactivation of Staphylococcus aureus ATCC 6538 which is an opportunistic pathogen of important relevance in the medical, pharmaceutical and food domains. Human blood plasma was chosen as the suspension medium because of its physiological importance in the transfusion field. It was shown that the optimization of all the selected parameters could lead to a high inactivation level (≈5log(10) decrease of the initial bacterial load) at a pressure level as low as 200MPa, underlining some synergistic effects among these parameters. Complete inactivation of the initial bacterial population was achieved for the following conditions: PR=50MPas(-1), AM=5×2min, T≈-5°C and P=300MPa.     

High hydrostatic pressure and biology: a brief history

Pressure as a thermodynamical parameter was successively introduced in physics, hydrometallurgy, geochemistry, and biology. In all cases, the main objective was to recreate a natural phenomenon (gas or liquid compressibility, synthesis or crystal growth of minerals, survival of deep sea microorganisms…). The introduction of high hydrostatic pressure (HHP) in Biology was an important scientific feature over the last hundred years. This paper describes the different steps that have led to the spreading of pressure in biology and the opening of new frontiers either in basic and applied researches due to the specific characteristics of the pressure parameter. Because of the low energy conveyed by this parameter, leading to the preservation of most organoleptic properties of foods, and its ability to inactivate many pathogens, the use of HHP began to spread at the end of the twentieth century into the food industry, in particular for the development of pathogen inactivation processes. Today, even if this field is still the first application domain for HHP, more and more research works have shown that this parameter could be of great interest in health and medicine sciences.     

Food processing by high hydrostatic pressure

High hydrostatic pressure (HHP) process, as a nonthermal process, can be used to inactivate microbes while minimizing chemical reactions in food. In this regard, a HHP level of 100 MPa (986.9 atm/1019.7 kgf/cm²) and more is applied to food. Conventional thermal process damages food components relating color, flavor, and nutrition via enhanced chemical reactions. However, HHP process minimizes the damages and inactivates microbes toward processing high quality safe foods. The first commercial HHP-processed foods were launched in 1990 as fruit products such as jams, and then some other products have been commercialized: retort rice products (enhanced water impregnation), cooked hams and sausages (shelf life extension), soy sauce with minimized salt (short-time fermentation owing to enhanced enzymatic reactions), and beverages (shelf life extension). The characteristics of HHP food processing are reviewed from viewpoints of nonthermal process, history, research and development, physical and biochemical changes, and processing equipment.     

Molecular Basis of the Behavior of Hepatitis A Virus Exposed to High Hydrostatic Pressure

Food-borne hepatitis A outbreaks may be prevented by subjecting foods at risk of virus contamination to moderate treatments of high hydrostatic pressure (HHP). A pretreatment promoting hepatitis A virus (HAV) capsid-folding changes enhances the virucidal effect of HHP, indicating that its efficacy depends on capsid conformation. HAV populations enriched in immature capsids (125S provirions) are more resistant to HHP, suggesting that mature capsids (150S virions) are more susceptible to this treatment. In addition, the monoclonal antibody (MAb) K24F2 epitope contained in the immunodominant site is a key factor for the resistance to HHP. Changes in capsid folding inducing a loss of recognition by MAb K24F2 render more susceptible conformations independently of the origin of such changes. Accordingly, codon usage-associated folding changes and changes stimulated by pH-dependent breathings, provided they confer a loss of recognition by MAb K24F2, induce a higher susceptibility to HHP. In conclusion, the resistance of HAV to HHP treatments may be explained by a low proportion of 150S particles combined with a good accessibility of the epitope contained in the immunodominant site close to the 5-fold axis.     

Biotechnological properties of distillery and laboratory yeasts in response to industrial stresses

The stress sensitivity of different wild-type strains was evaluated, as well as the response of cells arrested at different cell cycle positions to high hydrostatic pressure (HPP). HHP was chosen both for its importance in food decontamination and assessment of its suitability as a model for stress in general and understanding the yeast stress response. Studies were conducted with four industrial strains and four laboratory wild-type yeast strains (two haploid and two diploid) that differed in their backgrounds. Fundamental differences were found between the laboratory and industrial populations. Industrial strains were clearly more sensitive to hydrostatic pressure and ethanol stresses than the laboratory strains. However, ethanol production was higher in industrial strains than laboratory strains. Furthermore, no correlation was observed between ploidy and stress resistance. Yeast cells arrested in the G1 phase led to an enhancement in pressure tolerance compared to unarrested, G2 arrested, and S arrested cells. Moreover, cells arrested in the S phase were more sensitive to hydrostatic pressure than cells arrested in the G2 phase. Again, industrial strains were more sensitive than laboratory strains. HHP responses of industrial yeasts correlated well with both ethanol concentration and temperature stress, which suggests that it would be a useful model stress.     

Modeling conductive heat transfer and process uniformity during batch high-pressure processing of foods

A numerical model for predicting conductive heat transfer during batch high hydrostatic pressure (HHP) processing of foods was developed and tested for a food simulator (agar gel). For a comprehensive evaluation of the proposed method, both "conventional" HHP processes, HHP processes with gradual, step-by-step pressure buildup and pressure release, and pressure cycling HHP processes were included. In all cases, good agreement between experimental and predicted temperature profiles was observed. The model provides a very useful tool to evaluate batch HHP processes in terms of uniformity of any heat- and/or pressure-related effect. This is illustrated for inactivation of Bacillus subtilis alpha-amylase, an enzymatic model system with known pressure-temperature degradation kinetics.     

Spectrophotometric determination of folic acid in pharmaceutical preparations by coupling reactions with iminodibenzyl or 3-aminophenol or sodium molybdate-pyrocatechol

Novel coupling reagents are used for the simple and sensitive spectrophotometric determination of folic acid either in pure form or in its pharmaceutical preparations. The methods are based on the probable diazotization of the p-aminobenzoylglutamic acid obtained after reductive clevage of folic acid, followed by either coupling with iminodibenzyl to give a violet product with lambda(max) of 580nm or coupling with 3-aminophenol to produce an orange yellow-colored product with lambda(max) of 460nm. Sodium molybdate and pyrocatechol are used in the third method and the pale red-colored product formed has a lambda(max) of 490nm. The methods are highly reproducible and have been applied to the determination of folic acid in tablets and the results compare favorably with the official method. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods.     

Effect of pressure on islet amyloid polypeptide aggregation: revealing the polymorphic nature of the fibrillation process

Type II diabetes mellitus is a disease which is characterized by peripheral insulin resistance coupled with a progressive loss of insulin secretion that is associated with a decrease in pancreatic islet beta-cell mass and the deposition of amyloid in the extracellular matrix of beta-cells, which lead to islet cell death. The principal component of the islet amyloid is a pancreatic hormone called islet amyloid polypeptide (IAPP). High-pressure coupled with FT-IR spectroscopic and AFM studies were carried out to elucidate further information about the aggregation pathway as well as the aggregate structures of IAPP. To this end, a comparative fibrillation study of IAPP fragments was carried out as well. As high hydrostatic pressure (HHP) is acting to weaken or even prevent hydrophobic self-organization and electrostatic interactions, application of HHP has been used as a measure to reveal the importance of these interactions in the fibrillation process of IAPP and its fragments. IAPP preformed fibrils exhibit a strong polymorphism with heterogeneous structures, a large population of which are rather sensitive to high hydrostatic pressure, thus indicating a high percentage of ionic and hydrophobic interactions and loose packing of these species. Conversely, fragments 1-19 and 1-29 are resistant to pressure treatment, suggesting more densely packed aggregate structures with less void volume and strong cooperative hydrogen bonding. Furthermore, the FT-IR data indicate that fragment 1-29 has intermolecular beta-sheet conformational properties different from those of fragment 1-19, the latter exhibiting polymorphic behavior with more disordered structures and less strongly hydrogen bonded fibrillar assemblies. The data also suggest that hydrophobic interactions and/or less efficient packing of amino acids 30-37 region leads to the marked pressure sensitivity observed for full-length IAPP.     

Effect of domestic processing on bioactive compounds

Nowadays, most of the consumed foods are rarely ready for direct consumption. Food can be purchased at the local supermarket as fresh raw product such as meat, fruit, fish etc. or as manufactured product after an industrial processing (canned meat, dried fish, packed fruit etc.). But later on, both food types are usually submitted to culinary treatments which will transform the selected food into a cooked dish ready to eat. Domestic methods of food processing have been developed over the centuries to make the final product more attractive in flavour, appearance, taste and consistency. But, until the last centuries, none of the gourmets realize that at the same time, the cooking process was making their foods more digestible, microbiologically safer and more or less nutritive depending on the selected cooking technology. Besides consumer preferences, the selected cooking method is an important factor affecting not only the food chemical composition, but also the intake of bioactive compounds under normal dietary conditions. Therefore, in this work, the different culinary treatments and domestic cooking methods will be compared to define the optimal process to reduce the degradation of biologically active metabolites present in foods commonly consumed as an elaborated dish. Compounds such as carotenoids, glucosinolates, flavonoids and other phenolic compounds, ω-3 fatty acids, tocopherols, phytosterols, etc. have been pointed as bioactive compounds beneficial for human health. Apparently, they are able to prevent cardiovascular diseases (CVD), tumour formation, hiper-cholesterolemia in blood and other deleterious disorders. An adequate domestic practice might help to increase in taking of those functional molecules enhancing their functionality and reducing the risk of chronic diseases.     

Emergence and stability of high-pressure resistance in different food-borne pathogens

High hydrostatic pressure (HHP) processing is becoming a valuable nonthermal food pasteurization technique, although there is reasonable concern that bacterial HHP resistance could compromise the safety and stability of HHP-processed foods. While the degree of natural HHP resistance has already been shown to vary greatly among and within bacterial species, a still unresolved question remains as to what extent different food-borne pathogens can actually develop HHP resistance. In this study, we therefore examined and compared the intrinsic potentials for HHP resistance development among strains of Escherichia coli, Shigella flexneri, Salmonella enterica serovars Typhimurium and Enteritidis, Yersinia enterocolitica, Aeromonas hydrophila, Pseudomonas aeruginosa, and Listeria innocua using a selective enrichment approach. Interestingly, of all strains examined, the acquisition of extreme HHP resistance could be detected in only some of the E. coli strains, indicating that a specific genetic predisposition might be required for resistance development. Furthermore, once acquired, HHP resistance proved to be a very stable trait that was maintained for >80 generations in the absence of HHP exposure. Finally, at the mechanistic level, HHP resistance was not necessarily linked to derepression of the heat shock genes and was not related to the phenomenon of persistence.     

Effect of high hydrostatic pressure on hydration and activity of ribozymes

Formation and stabilization of RNA structure in the cell depends on its interaction with solvent and metal ions. High hydrostatic pressure (HHP) is a convenient tool in an analysis of the role of small molecules in the structure stabilization of biological macromolecules. Analysis of HHP effect and various concentrations of ions showed that water induce formation of the active ribozyme structure. So, it is clear that water is the driving force of conformational changes of nucleic acid.     

Foods for patients with celiac disease.

As a general rule patients with celiac disease must avoid five cereals--wheat rye, triticale, barley and oats. Very sensitive individuals must also avoid two products of these cereals--malt and hydrolyzed vegetable protein. Some less sensitive individuals may be able to tolerate barley and oats in small quantities. All other foods are acceptable, including the cereals corn, rice, buckwheat, millet and sorghum, as well as malt-flavored breakfast cereals. Wine, spirits, beer and ale are also acceptable unless otherwise contraindicated. Monosodium glutamate, other food additives and pharmaceutical preparations are also acceptable. The ingredients of prepackaged processed foods are listed on the labels. Patients with celiac disease must examine labels to ensure that they avoid the harmful cereals. With appropriate precautions they need not be concerned about eating away from home.     

Highly sensitive reaction of tryptophan with p-phenylenediamine

A simple and sensitive spectrophotometric method for the determination of tryptophan (TRP) is described. The method is based on the coupling reaction of tryptophan with diazotized p-phenylenediamine dihydrochloride (PPDD) in sulfuric acid medium to give the colored product having an absorption maximum at 520 nm. The coupled product was stable for 2h. Beer's law is obeyed in the tryptophan concentration range of 0.25-11 microg/ml. The method is applied for the analysis of pharmaceutical preparations of tryptophan and also in protein samples for tryptophan. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed method and the significant feature of the method is that most of the amino acids do not interfere in the determination of tryptophan.     

Biotransformation studies using hairy root cultures - A review

Agrobacterium rhizogenes induced hairy root cultures are entering into a new juncture of functional research in generating pharmaceutical lead compounds by bringing about chemical transformations aided through its inherent enzyme resources. Rational utilization of hairy root cultures as highly effective biotransformation systems has come into existence in the last twenty years involving a wide range of plant systems as well as exogenous substrates and diverse chemical reactions. To date, hairy root cultures are preferred over plant cell/callus and suspension cultures as biocatalyst due to their genetic/biochemical stability, hormone-autotrophy, multi-enzyme biosynthetic potential mimicking that of the parent plants and relatively low-cost cultural requirements. The resultant biotransformed molecules, that are difficult to make by synthetic organic chemistry, can unearth notable practical efficacies by acquiring improved physico-chemical properties, bioavailability, lower toxicity and broader therapeutic properties. The present review summarizes the overall reported advances made in the area of hairy root mediated biotransformation of exogenous substrates with regard to their reaction types, plant systems associated, bacterial strains/molecules involved and final product recovery.     

超高压对植物乳杆菌能量代谢影响的研究

以植物乳杆菌ATCC8014为试材,研究超高压对其能量代谢的影响。建立了用氯化碘硝基四唑紫测定ATCC8014的INT代谢还原活性的比色法。用比色法测定了超高压对ATCC8014的INT代谢还原活性与葡萄糖利用的影响。试验结果表明,150~250MPa作用15min在MRS琼脂培养基上随着压力的增大菌落数显著降低,INT代谢还原活性降低显著,葡萄糖的利用变化不明显;超过300MPa后,葡萄糖的利用才显著降低;400MPa处理15min,尽管在MRS琼脂培养基上菌落数低于检测限,INT代谢还原活性为0%,而葡萄糖的利用能力仍为对照组的56.1%,超高压作用下ATCC8014的灭活与INT代谢还原活性的降低的相关性较好。说明ATCC8014的细胞膜上参与葡萄糖的吸收和运输的酶、糖酵解的酶与调节系统比三羧酸循环的酶与调节系统较耐压。三羧酸循环比糖酵解对超高压敏感,三羧酸循环的抑制是超高压灭活其的重要原因,这为了探讨超高压杀灭植物乳杆菌的机制提供了一定的理论依据。     

Application of Ultra-Centrifugation and Bench-Top <Superscript>19</Superscript>F NMR for Measuring Drug Phase Partitioning for the Ophthalmic Oil-in-Water Emulsion Products

Generic drug products are expected to have the same active pharmaceutical ingredient (API) (Q1) with the same content (Q2) and microstructure arrangement (Q3) as the innovator product. In complex oil-in-water emulsion drugs, the hydrophobic API is mainly formulated in oil droplets stabilized by surfactant and micelles composed of extra surfactant molecules. The API phase partition in oil and water (mainly micelle) is a critical quality attribute (CQA) of emulsion product in demonstrating physicochemical equivalence using difluprednate (DFPN) emulsion product Durezol® as a model, we developed a novel low-field benchtop NMR method to demonstrate its applicability in measuring DFPN phase partition for ophthalmic oil-in-water emulsion products. Low-field ¹⁹F spectra were collected for DFPN in formulation, in water phase and oil phase after separation from ultra-centrifugation. The NMR data showed the mass balance of DFPN before and after phase separation. The average water phase content of different Durezol® lots was 32 ± 3% with 1% variation from method reproducibility test. The partition results were 52 ± 2% for the in-house control products prepared in Q1/Q2 equivalence to Durezol® but by a different process. The significant difference in DFPN-phase partition between Durezol® and the in-house formulation demonstrated manufacture difference readily changed the API partition. The newly developed ultra-centrifugation and ¹⁹F NMR by benchtop instrument is a simple, robust, and sensitive analytical method for ophthalmic emulsion drug product development and control.     

Inactivation Effects on Proteins in a Needle‐free Vaccine Injector

Needle‐free powder injectors using a supersonic gas jet to force powder particles into the skin are suitable devices for the reliable delivery of powdered vaccines into viable epidermis. The peculiarities of the injection mechanism lead to a more comfortable injection for the patient, but in return to an increase in the stress on the pharmaceutical formulation in comparison to conventional injections with needle and syringe. As a lot of the conceivable drugs for needle‐free powder injection, particularly protein vaccines, are very sensitive bio‐molecules with an extremely fragile molecular structure, their resilience to the potential molecule damaging processes during the actuation with the device and the preservation of their biological activity are the crucial factors. Therefore it was the central aim of this work to identify and investigate the reasons for the loss of biological activity. The imaginable responsible process steps were investigated separately and the amounts of activity loss related to them. The manufacture of the powder and the loading of the cassette containing the formulation are examined. Moreover, the effects of static pressure exposition, the stress occurring because of the particle transport through the injector device, inevitably accompanied by impacts and shear stress and the influence of the particle impact with the target, were the focus of this investigation. Furthermore, different formulations of protein in combination with pharmaceutical excipients well‐known for their protein stabilizing effects during the powder manufacture process were examined in regard to their potential suitability to protect protein against the stress occurring throughout the needle‐free injection.     

A possible involvement of virus-associated protease in the fusion of Sendai virus envelopes with human erythrocytes

A proteolytic activity is shown to be associated with relatively purified preparations of intact Sendai virus particles or with their reconstituted envelopes which are vesicles containing mainly the viral glycoproteins. Intact Sendai virus as well as reconstituted Sendai virus envelopes have been shown to be able to hydrolyze various protein molecules such as the human erythrocyte membrane polypeptide designated as band 3 and soluble polypeptides such as histone and insulin B-chain. The results of the present work raise the possibility that a direct correlation exists between the virus-associated proteolytic activity and the ability of the virions to lyse cells, to fuse with their membranes, and to promote cell-cell fusion. Inhibitors of proteolytic enzymes such as phenylmethylsulfonyl fluoride, tosyllysinechloromethylketone and tosylamidephenylethylchloromethylketone, or combinations thereof, inhibit the virus-associated proteolytic activity concomitantly with inhibition of its hemolytic and fusogenic activities. Electron microscopic studies showed that the various inhibitors did not affect the binding ability of the virus preparations. The possible involvement of a protease in the process of virus-membrane fusion is discussed.     

Value in global standardization of outbred rats for regulatory pharmaceuticals studies.

As a result of a revolution in globalization over the last 10 years, few pharmaceutical companies conduct their non-clinical studies within a single region and virtually none conduct all non-clinical studies in house. The increased activity of product in-licensing at all stages of development results in further segmentation of toxicological source information. Juxtaposed to this segmental collection of toxicology information is the nature of toxicological testing for pharmaceuticals, an iterative process, carefully building subsequent testing designs and analyses upon the foundation of previously identified outcomes and associated safety issues. As a consequence of the segmentation of global product development, today's analyses of toxicological outcomes for single pharmaceutical projects often involve a conglomeration of results from studies conducted in several world regions using a variety of sources of animals. Such practices can lead to discordant study results and difficulty in understanding or rationalizing a compound's toxicological and pharmacological profile, and eventually how this relates to human risk. Despite the variety in source information, an integration of the total toxicology/pharmacology data must be made by industry, and/or by health authorities. To improve this integration, the development and use of better standardized genetics for rodent species used in pharmaceutical testing is more imperative today than in the past, as a result of a constellation of changes in industry policies.     

Process development in the QbD paradigm: Role of process integration in process optimization for production of biotherapeutics

Biotherapeutics have become the focus of the pharmaceutical industry due to their proven effectiveness in managing complex diseases. Downstream processes of these molecules consist of several orthogonal, high resolution unit operations designed so as to be able to separate variants having very similar physicochemical properties. Typical process development involves optimization of the individual unit operations based on Quality by Design principles in order to define the design space within which the process can deliver product that meets the predefined specifications. However, limited efforts are dedicated to understanding the interactions between the unit operations. This paper aims to showcase the importance of understanding these interactions and thereby arrive at operating conditions that are optimal for the overall process. It is demonstrated that these are not necessarily same as those obtained from optimization of the individual unit operations. Purification of Granulocyte Colony Stimulating Factor (G‐CSF), a biotherapeutic expressed in E. coli., has been used as a case study. It is evident that the suggested approach results in not only higher yield (91.5 vs. 86.4) but also improved product quality (% RP‐HPLC purity of 98.3 vs. 97.5) and process robustness. We think that this paper is very relevant to the present times when the biotech industry is in the midst of implementing Quality by Design towards process development. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:355–362, 2016